Set2 and H3K36 regulate the Drosophila male X chromosome in a context-specific manner, independent from MSL complex spreading.

Dosage compensation in Drosophila involves upregulating male X-genes two-fold. This process is carried out by the MSL (male-specific lethal) complex, which binds high-affinity sites and spreads to surrounding genes. Current models of MSL spreading focus on interactions betwen MSL3 (male-specific lethal 3) and Set2-dependent histone marks like trimethylated H3 lysine-36 (H3K36me3). However, Set2 could affect DC via another target, or there could be redundancy between canonical H3.2 and variant H3.3 histones. Furthermore, it is important to parse male-specific effects from those that are X-specific. To discriminate among these possibilities, we employed genomic approaches in H3K36 'residue' and Set2 'writer' mutants. The results confirm a role for Set2 in X-gene regulation, but show that expression trends in males are often mirrored in females. Instead of global, male-specific reduction of X-genes in Set2 or H3K36 mutants, we observe heterogeneous effects. Interestingly, we identified groups of differentially expressed genes (DEGs) whose changes were in opposite directions following loss of H3K36 or Set2, suggesting that H3K36me states have reciprocal functions. In contrast to H4K16R controls, differential expression analysis of combined H3.2K36R/H3.3K36R mutants showed neither consistent reduction in X-gene expression, nor correlation with MSL3 binding. Motif analysis of the DEGs implicated BEAF-32 and other insulator proteins in Set2/H3K36-dependent regulation. Overall, the data are inconsistent with the prevailing model wherein H3K36me3 is essential for spreading the MSL complex to genes along the male X. Rather, we propose that Set2 and H3K36 support DC indirectly, via processes that are utilized by MSL but common to both sexes.

NUP98 fusion proteins and KMT2A-MENIN antagonize PRC1.1 to drive gene expression in AML.

Control of stem cell-associated genes by Trithorax group (TrxG) and Polycomb group (PcG) proteins is frequently misregulated in cancer. In leukemia, oncogenic fusion proteins hijack the TrxG homolog KMT2A and disrupt PcG activity to maintain pro-leukemogenic gene expression, though the mechanisms by which oncofusion proteins antagonize PcG proteins remain unclear. Here, we define the relationship between NUP98 oncofusion proteins and the non-canonical polycomb repressive complex 1.1 (PRC1.1) in leukemia using Menin-KMT2A inhibitors and targeted degradation of NUP98 fusion proteins. Eviction of the NUP98 fusion-Menin-KMT2A complex from chromatin is not sufficient to silence pro-leukemogenic genes. In the absence of PRC1.1, key oncogenes remain transcriptionally active. Transition to a repressed chromatin state requires the accumulation of PRC1.1 and repressive histone modifications. We show that PRC1.1 loss leads to resistance to small-molecule Menin-KMT2A inhibitors in vivo. Therefore, a critical function of oncofusion proteins that hijack Menin-KMT2A activity is antagonizing repressive chromatin complexes.

KMT2D-mediated H3K4me1 recruits YBX1 to facilitate triple-negative breast cancer progression through epigenetic activation of c-Myc.

BACKGROUND: Lysine methyltransferase 2D (KMT2D) mediates mono-methylation of histone H3 lysine 4 (H3K4me1) in mammals. H3K4me1 mark is involved in establishing an active chromatin structure to promote gene transcription. However, the precise molecular mechanism underlying the KMT2D-mediated H3K4me1 mark modulates gene expression in triple-negative breast cancer (TNBC) progression is unresolved. METHODS AND RESULTS: We recognized Y-box-binding protein 1 (YBX1) as a "reader" of the H3K4me1 mark, and a point mutation of YBX1 (E121A) disrupted this interaction. We found that KMT2D and YBX1 cooperatively promoted cell growth and metastasis of TNBC cells in vitro and in vivo. The expression levels of KMT2D and YBX1 were both upregulated in tumour tissues and correlated with poor prognosis for breast cancer patients. Combined analyses of ChIP-seq and RNA-seq data indicated that YBX1 was co-localized with KMT2D-mediated H3K4me1 in the promoter regions of c-Myc and SENP1, thereby activating their expressions in TNBC cells. Moreover, we demonstrated that YBX1 activated the expressions of c-Myc and SENP1 in a KMT2D-dependent manner. CONCLUSION: Our results suggest that KMT2D-mediated H3K4me1 recruits YBX1 to facilitate TNBC progression through epigenetic activation of c-Myc and SENP1. These results together unveil a crucial interplay between histone mark and gene regulation in TNBC progression, thus providing novel insights into targeting the KMT2D-H3K4me1-YBX1 axis for TNBC treatment. HIGHLIGHTS: YBX1 is a KMT2D-mediated H3K4me1-binding effector protein and mutation of YBX1 (E121A) disrupts its binding to H3K4me1. KMT2D and YBX1 cooperatively promote TNBC proliferation and metastasis by activating c-Myc and SENP1 expression in vitro and in vivo. YBX1 is colocalized with H3K4me1 in the c-Myc and SENP1 promoter regions in TNBC cells and increased YBX1 expression predicts a poor prognosis in breast cancer patients.

Lysine methyltransferase 2 plays a key role in the encystation process in the parasite Giardia lamblia.

Histone post-translational modifications are extensively studied for their role in regulating gene transcription and cellular environmental adaptation. Research into these modifications has recently begun in the protozoan parasite Giardia lamblia, focusing on histone-modifying enzymes and specific post-translational changes. In the transformation from the trophozoite to the cyst form in the life cycle of this parasite, significant morphological and genetic alterations occur, culminating in the synthesis of cyst wall proteins responsible for forming the protective cyst wall. It has been previously demonstrated that histone deacetylation is required during encystation and that the enzyme lysine methyltransferase 1 is involved in the upregulation of encystation. Our study aims to extend the analysis to lysine methyltransferase 2 (GlKMT2) function. For this, two constructs were generated: one that downregulate the expression of GLKMT2 via antisense (glkmt2-as transgenic cells) and the other overexpressing GlKMT2 (glkmt2-ha transgenic cells). We found that the glktm2-as transgenic cells showed an arrest in progress at the late encystation stage. Consequently, the number of cysts produced was lower than that of the control cells. On the other hand, we found that the overexpression of GlKMT2 acts as a negative mutant of the enzyme. In this way, these glktm2-ha transgenic cells showed the same behavior during growth and encystation as glkmt2-as transgenic cells. This interplay between different enzymes acting during encystation reveals the complex process behind the differentiation of the parasite. Understanding how these enzymes play their role during the encystation of the parasite would allow the design of inhibitors to control the parasite.

Ketogenic diet modifies ribosomal protein dysregulation in KMT2D Kabuki syndrome.

BACKGROUND: Kabuki syndrome (KS) is a genetic disorder caused by DNA mutations in KMT2D, a lysine methyltransferase that methylates histones and other proteins, and therefore modifies chromatin structure and subsequent gene expression. Ketones, derived from the ketogenic diet, are histone deacetylase inhibitors that can 'open' chromatin and encourage gene expression. Preclinical studies have shown that the ketogenic diet rescues hippocampal memory neurogenesis in mice with KS via the epigenetic effects of ketones. METHODS: Single-cell RNA sequencing and mass spectrometry-based proteomics were used to explore molecular mechanisms of disease in individuals with KS (n = 4) versus controls (n = 4). FINDINGS: Pathway enrichment analysis indicated that loss of function mutations in KMT2D are associated with ribosomal protein dysregulation at an RNA and protein level in individuals with KS (FDR <0.05). Cellular proteomics also identified immune dysregulation and increased abundance of other lysine modification and histone binding proteins, representing a potential compensatory mechanism. A 12-year-old boy with KS, suffering from recurrent episodes of cognitive decline, exhibited improved cognitive function and neuropsychological assessment performance after 12 months on the ketogenic diet, with concomitant improvement in transcriptomic ribosomal protein dysregulation. INTERPRETATION: Our data reveals that lysine methyltransferase deficiency is associated with ribosomal protein dysfunction, with secondary immune dysregulation. Diet and the production of bioactive molecules such as ketone bodies serve as a significant environmental factor that can induce epigenetic changes and improve clinical outcomes. Integrating transcriptomic, proteomic, and clinical data can define mechanisms of disease and treatment effects in individuals with neurodevelopmental disorders. FUNDING: This study was supported by the Dale NHMRC Investigator Grant (APP1193648) (R.D), Petre Foundation (R.D), and The Sydney Children's Hospital Foundation/Kids Research Early and Mid-Career Researcher Grant (E.T).

Set2 and H3K36 regulate the Drosophila male X chromosome in a context-specific manner, independent from MSL complex spreading.

Dosage compensation in Drosophila involves upregulating male X-genes two-fold. This process is carried out by the MSL (male-specific lethal) complex, which binds high-affinity sites and spreads to surrounding genes. Current models of MSL spreading focus on interactions of MSL3 (male-specific lethal 3) with histone marks; in particular, Set2-dependent H3 lysine-36 trimethylation (H3K36me3). However, Set2 might affect DC via another target, or there could be redundancy between canonical H3.2 and variant H3.3 histones. Further, it is difficult to parse male-specific effects from those that are simply X-specific. To discriminate among these possibilities, we employed genomic approaches in H3K36 (residue) and Set2 (writer) mutants. The results confirm a role for Set2 in X-gene regulation, but show that expression trends in males are often mirrored in females. Instead of global male-specific reduction of X-genes in Set2/H3K36 mutants, the effects were heterogeneous. We identified cohorts of genes whose expression was significantly altered following loss of H3K36 or Set2, but the changes were in opposite directions, suggesting that H3K36me states have reciprocal functions. In contrast to H4K16R controls, analysis of combined H3.2K36R/H3.3K36R mutants neither showed consistent reduction in X-gene expression, nor any correlation with MSL3 binding. Examination of other developmental stages/tissues revealed additional layers of context-dependence. Our studies implicate BEAF-32 and other insulator proteins in Set2/H3K36-dependent regulation. Overall, the data are inconsistent with the prevailing model wherein H3K36me3 directly recruits the MSL complex. We propose that Set2 and H3K36 support DC indirectly, via processes that are utilized by MSL but common to both sexes.

Knocking Out Chloroplastic Aldolases/Rubisco Lysine Methyltransferase Enhances Biomass Accumulation in Nannochloropsis oceanica under High-Light Stress.

Rubisco large-subunit methyltransferase (LSMT), a SET-domain protein lysine methyltransferase, catalyzes the formation of trimethyl-lysine in the large subunit of Rubisco or in fructose-1,6-bisphosphate aldolases (FBAs). Rubisco and FBAs are both vital proteins involved in CO2 fixation in chloroplasts; however, the physiological effect of their trimethylation remains unknown. In Nannochloropsis oceanica, a homolog of LSMT (NoLSMT) is found. Phylogenetic analysis indicates that NoLSMT and other algae LSMTs are clustered in a basal position, suggesting that algal species are the origin of LSMT. As NoLSMT lacks the His-Ala/ProTrp triad, it is predicted to have FBAs as its substrate instead of Rubisco. The 18-20% reduced abundance of FBA methylation in NoLSMT-defective mutants further confirms this observation. Moreover, this gene (nolsmt) can be induced by low-CO2 conditions. Intriguingly, NoLSMT-knockout N. oceanica mutants exhibit a 9.7-13.8% increase in dry weight and enhanced growth, which is attributed to the alleviation of photoinhibition under high-light stress. This suggests that the elimination of FBA trimethylation facilitates carbon fixation under high-light stress conditions. These findings have implications in engineering carbon fixation to improve microalgae biomass production.

Lysine Methyltransferase 9 (KMT9) Is an Actionable Target in Muscle-Invasive Bladder Cancer.

Novel treatment modalities are imperative for the challenging management of muscle-invasive and metastatic BC to improve patient survival rates. The recently identified KMT9, an obligate heterodimer composed of KMT9α and KMT9ß, regulates the growth of various types of tumors such as prostate, lung, and colon cancer. While the overexpression of KMT9α was previously observed to be associated with aggressive basal-like MIBC in an analysis of patients' tissue samples, a potential functional role of KMT9 in this type of cancer has not been investigated to date. In this study, we show that KMT9 regulates proliferation, migration, and invasion of various MIBC cell lines with different genetic mutations. KMT9α depletion results in the differential expression of genes regulating the cell cycle, cell adhesion, and migration. Differentially expressed genes include oncogenes such as EGFR and AKT1 as well as mediators of cell adhesion or migration such as DAG1 and ITGA6. Reduced cell proliferation upon KMT9α depletion is also observed in Pten/Trp53 knockout bladder tumor organoids, which cannot be rescued with an enzymatically inactive KMT9α mutant. In accordance with the idea that the catalytic activity of KMT9 is required for the control of cellular processes in MIBC, a recently developed small-molecule inhibitor of KMT9 (KMI169) also impairs cancer cell proliferation. Since KMT9α depletion also restricts the growth of xenografts in mice, our data suggest that KMT9 is an actionable novel therapeutic target for the treatment of MIBC.

Approaching the catalytic mechanism of protein lysine methyltransferases by biochemical and simulation techniques.

Protein lysine methyltransferases (PKMTs) transfer up to three methyl groups to the side chains of lysine residues in proteins and fulfill important regulatory functions by controlling protein stability, localization and protein/protein interactions. The methylation reactions are highly regulated, and aberrant methylation of proteins is associated with several types of diseases including neurologic disorders, cardiovascular diseases, and various types of cancer. This review describes novel insights into the catalytic machinery of various PKMTs achieved by the combined application of biochemical experiments and simulation approaches during the last years, focusing on clinically relevant and well-studied enzymes of this group like DOT1L, SMYD1-3, SET7/9, G9a/GLP, SETD2, SUV420H2, NSD1/2, different MLLs and EZH2. Biochemical experiments have unraveled many mechanistic features of PKMTs concerning their substrate and product specificity, processivity and the effects of somatic mutations observed in PKMTs in cancer cells. Structural data additionally provided information about the substrate recognition, enzyme-substrate complex formation, and allowed for simulations of the substrate peptide interaction and mechanism of PKMTs with atomistic resolution by molecular dynamics and hybrid quantum mechanics/molecular mechanics methods. These simulation technologies uncovered important mechanistic details of the PKMT reaction mechanism including the processes responsible for the deprotonation of the target lysine residue, essential conformational changes of the PKMT upon substrate binding, but also rationalized regulatory principles like PKMT autoinhibition. Further developments are discussed that could bring us closer to a mechanistic understanding of catalysis of this important class of enzymes in the near future. The results described here illustrate the power of the investigation of enzyme mechanisms by the combined application of biochemical experiments and simulation technologies.

Impact of Histone Lysine Methyltransferase SUV4-20H2 on Cancer Onset and Progression with Therapeutic Potential.

Histone lysine methyltransferase SUV4-20H2, a member of the suppressor of variegation 4-20 homolog (SUV4-20) family, has a critical impact on the regulation of chromatin structure and gene expression. This methyltransferase establishes the trimethylation of histone H4 lysine 20 (H4K20me3), a repressive histone mark that affects several cellular processes. Deregulated SUV4-20H2 activity has been associated with altered chromatin dynamics, leading to the misregulation of key genes involved in cell cycle control, apoptosis and DNA repair. Emerging research evidence indicates that SUV4-20H2 acts as a potential epigenetic modifier, contributing to the development and progression of several malignancies, including breast, colon and lung cancer, as well as renal, hepatocellular and pancreatic cancer. Understanding the molecular mechanisms that underlie SUV4-20H2-mediated effects on chromatin structure and gene expression may provide valuable insights into novel therapeutic strategies for targeting epigenetic alterations in cancer. Herein, we discuss structural and functional aspects of SUV4-20H2 in cancer onset, progression and prognosis, along with current targeting options.

Genome-wide identification of histone lysine methyltransferases and their implications in the epigenetic regulation of eggshell formation-related genes in a trematode parasite Clonorchis sinensis.

Epigenetic writers including DNA and histone lysine methyltransferases (DNMT and HKMT, respectively) play an initiative role in the differentiation and development of eukaryotic organisms through the spatiotemporal regulation of functional gene expressions. However, the epigenetic mechanisms have long been suspected in helminth parasites lacking the major DNA methyltransferases DNMT1 and DNMT3a/3b. Very little information on the evolutionary status of the epigenetic tools and their role in regulating chromosomal genes is currently available in the parasitic trematodes. We previously suggested the probable role of a DNMT2-like protein (CsDNMT2) as a genuine epigenetic writer in a trematode parasite Clonorchis sinensis. Here, we analyzed the phylogeny of HKMT subfamily members in the liver fluke and other platyhelminth species. The platyhelminth genomes examined conserved genes for the most of SET domain-containing HKMT and Disruptor of Telomeric Silencing 1 subfamilies, while some genes were expanded specifically in certain platyhelminth genomes. Related to the high gene dosages for HKMT activities covering differential but somewhat overlapping substrate specificities, variously methylated histones were recognized throughout the tissues/organs of C. sinensis adults. The temporal expressions of genes involved in eggshell formation were gradually decreased to their lowest levels proportionally to aging, whereas those of some epigenetic tool genes were re-boosted in the later adult stages of the parasite. Furthermore, these expression levels were significantly affected by treatment with DNMT and HKMT inhibitors. Our data strongly suggest that methylated histones are potent epigenetic markers that modulate the spatiotemporal expressions of C. sinensis genes, especially those involved in sexual reproduction.

Lysine methyltransferase 5C increases the proliferation and metastatic abilities of clear cell renal cell carcinoma via aerobic glycolysis.

Among all types of renal cancer, clear cell renal cell carcinoma (ccRCC) is the most common and lethal subtype and is associated with a high risk of metastasis and recurrence. Histone modifications regulate several biological processes that are fundamental to the development of cancer. Lysine methyltransferase 5C (KMT5C; also known as SUV420H2) is an epigenetic modifier responsible for the trimethylation of H4K20, which drives critical cellular events, including genome integrity, cell growth and epithelial­mesenchymal transition (EMT), in various types of cancer. However, the role of KMT5C in ccRCC remains unclear. As such, the expression and function of KMT5C in ccRCC were investigated in the present study. KMT5C expression was significantly increased in ccRCC tissues compared with normal tissues (P<0.0001), and it was closely associated with the overall survival rate of patients with ccRCC. By establishing ccRCC cell lines with KMT5C expression knockdown, the role of KMT5C in the maintenance of aerobic glycolysis in ccRCC cells via the regulation of several vital glycolytic genes was identified. Additionally, KMT5C promoted the proliferation and EMT of ccRCC cells by controlling crucial EMT transcriptional factors. Together, these data suggested that KMT5C may act as an oncoprotein, guide molecular diagnosis, and shed light on novel drug development and therapeutic strategies for patients with ccRCC.

Regulation of adipogenesis by histone methyltransferases.

Epigenetic regulation is a critical component of lineage determination. Adipogenesis is the process through which uncommitted stem cells or adipogenic precursor cells differentiate into adipocytes, the most abundant cell type of the adipose tissue. Studies examining chromatin modification during adipogenesis have provided further understanding of the molecular blueprint that controls the onset of adipogenic differentiation. Unlike histone acetylation, histone methylation has context dependent effects on the activity of a transcribed region of DNA, with individual or combined marks on different histone residues providing distinct signals for gene expression. Over half of the 42 histone methyltransferases identified in mammalian cells have been investigated in their role during adipogenesis, but across the large body of literature available, there is a lack of clarity over potential correlations or emerging patterns among the different players. In this review, we will summarize important findings from studies published in the past 15 years that have investigated the role of histone methyltransferases during adipogenesis, including both protein arginine methyltransferases (PRMTs) and lysine methyltransferases (KMTs). We further reveal that PRMT1/4/5, H3K4 KMTs (MLL1, MLL3, MLL4, SMYD2 and SET7/9) and H3K27 KMTs (EZH2) all play positive roles during adipogenesis, while PRMT6/7 and H3K9 KMTs (G9a, SUV39H1, SUV39H2, and SETDB1) play negative roles during adipogenesis.

KMT2D preferentially binds mRNAs of the genes it regulates, suggesting a role in RNA processing.

Histone lysine methyltransferases (HKMTs) perform vital roles in cellular life by controlling gene expression programs through the posttranslational modification of histone tails. Since many of them are intimately involved in the development of different diseases, including several cancers, understanding the molecular mechanisms that control their target recognition and activity is vital for the treatment and prevention of such conditions. RNA binding has been shown to be an important regulatory factor in the function of several HKMTs, such as the yeast Set1 and the human Ezh2. Moreover, many HKMTs are capable of RNA binding in the absence of a canonical RNA binding domain. Here, we explored the RNA binding capacity of KMT2D, one of the major H3K4 monomethyl transferases in enhancers, using RNA immunoprecipitation followed by sequencing. We identified a broad range of coding and non-coding RNAs associated with KMT2D and confirmed their binding through RNA immunoprecipitation and quantitative PCR. We also showed that a separated RNA binding region within KMT2D is capable of binding a similar RNA pool, but differences in the binding specificity indicate the existence of other regulatory elements in the sequence of KMT2D. Analysis of the bound mRNAs revealed that KMT2D preferentially binds co-transcriptionally to the mRNAs of the genes under its control, while also interacting with super enhancer- and splicing-related non-coding RNAs. These observations, together with the nuclear colocalization of KMT2D with differentially phosphorylated forms of RNA Polymerase II suggest a so far unexplored role of KMT2D in the RNA processing of the nascent transcripts.

Integration of ligand and structure-based pharmacophore screening for the identification of novel natural leads against Euchromatic histone lysine methyltransferase 2 (EHMT2/G9a).

Herein, we report a blended ligand and structure-based pharmacophore screening approach to identify new natural leads against the Protein Lysine Methyltransferase 2 (EHMT2/G9a). The EHMT2/G9a has been associated with Cancer, Alzheimer's, and aging and is considered an emerging drug target having no clinically passed inhibitor. Purposefully, we developed the ligand-based pharmacophore (Pharmacophore-L) based on the common features of known inhibitors and the structure-based pharmacophore (Pharmacophore-S) based on the interaction profile of available crystal structures. The Pharmacophore-L and Pharmacophore-S were subjected to multiple tiers of validations and utilized in combination for the screening of total 741543 compounds coming from multiple databases. Additional layers of stringency were applied in the screening process to test drug-likeness (using Lipinski's rule, Veber's rule, SMARTS and ADMET filtration), to rule out any toxicity (TOPKAT analysis). The interaction profiles, stabilities, and comparative analysis against the reference were carried out by flexible docking, MD simulation, and MM-GBSA analysis, which finally led to three leads as potential inhibitors of G9a.Communicated by Ramaswamy H. Sarma.

In Vivo and In Vitro Characterization of the RNA Binding Capacity of SETD1A (KMT2F).

For several histone lysine methyltransferases (HKMTs), RNA binding has been already shown to be a functionally relevant feature, but detailed information on the RNA interactome of these proteins is not always known. Of the six human KMT2 proteins responsible for the methylation of the H3K4 residue, two-SETD1A and SETD1B-contain RNA recognition domains (RRMs). Here we investigated the RNA binding capacity of SETD1A and identified a broad range of interacting RNAs within HEK293T cells. Our analysis revealed that similar to yeast Set1, SETD1A is also capable of binding several coding and non-coding RNAs, including RNA species related to RNA processing. We also show direct RNA binding activity of the individual RRM domain in vitro, which is in contrast with the RRM domain found in yeast Set1. Structural modeling revealed important details on the possible RNA recognition mode of SETD1A and highlighted some fundamental differences between SETD1A and Set1, explaining the differences in the RNA binding capacity of their respective RRMs.

Rare de novo gain-of-function missense variants in DOT1L are associated with developmental delay and congenital anomalies.

Misregulation of histone lysine methylation is associated with several human cancers and with human developmental disorders. DOT1L is an evolutionarily conserved gene encoding a lysine methyltransferase (KMT) that methylates histone 3 lysine-79 (H3K79) and was not previously associated with a Mendelian disease in OMIM. We have identified nine unrelated individuals with seven different de novo heterozygous missense variants in DOT1L through the Undiagnosed Disease Network (UDN), the SickKids Complex Care genomics project, and GeneMatcher. All probands had some degree of global developmental delay/intellectual disability, and most had one or more major congenital anomalies. To assess the pathogenicity of the DOT1L variants, functional studies were performed in Drosophila and human cells. The fruit fly DOT1L ortholog, grappa, is expressed in most cells including neurons in the central nervous system. The identified DOT1L variants behave as gain-of-function alleles in flies and lead to increased H3K79 methylation levels in flies and human cells. Our results show that human DOT1L and fly grappa are required for proper development and that de novo heterozygous variants in DOT1L are associated with a Mendelian disease.

(R)-PFI-2 Analogues as Substrates and Inhibitors of Histone Lysine Methyltransferase SETD7.

(R)-PFI-2 is a histone substrate-competitive inhibitor of the human histone lysine monomethyltransferase SETD7. Aimed at developing potent inhibitors of SETD7 that can also act as small molecule substrates, we replaced the pyrrolidine ring of (R)-PFI-2 with several side chains bearing nucleophilic functional groups. We explored the inhibitory activity of 20 novel (R)-PFI-2 analogues, and found that the most potent analogue has a hydroxyethyl side chain (7). SETD7's ability to catalyse methylation of (R)-PFI-2-based small molecules was evaluated by mass spectrometric assays, and we observed efficient methylation of analogues bearing lysine mimicking nucleophilic amines. The optimal side chain was found to be an aminoethyl group (1), which was surprisingly also dimethylated by SETD7. The work demonstrates that small molecules can act as both substrates and inhibitors of biomedically important SETD7.

Pharmacological inhibition of the mixed lineage leukemia 1-menin interaction aggravates acute kidney injury induced by folic acid and ischemia-reperfusion in mice.

Mixed lineage leukemia 1 (MLL1) is a methyltransferase that induces histone H3 lysine 4 trimethylation (H3K4me3) and partially exerts its untoward functional effects by interacting with multiple subunits including menin and WD repeat-containing protein 5 (WDR5). In this study, we investigated the role and mechanisms of MLL1 in murine models of acute kidney injury induced by folic acid (FA) and ischemia-reperfusion. Injury to the kidney elevated the expression of MLL1, menin, WDR5, and H3K4Me3, which was accompanied by increased serum creatinine and blood urea nitrogen, renal tubular injury, and apoptosis. Pharmacological inhibition of MLL1 activity with MI503 to disrupt the interaction between MLL1 with menin further increased serum creatinine and blood urea nitrogen levels, enhanced expression of neutrophil gelatinase-associated lipocalin and kidney injury molecule-1, and induced more apoptosis in the kidney following FA and ischemia-reperfusion injury. In contrast, MI503 treatment decreased the expression of vimentin and proliferating cell nuclear antigens. Similarly, treatment with MM102 to disrupt the interaction between MLL1 and WDR5 also worsened renal dysfunction, aggravated tubular cell injury, increased apoptosis, and inhibited cellular dedifferentiation and proliferation in mice following FA injection. Moreover, MI503 inhibited FA-induced phosphorylation of epidermal growth factor receptor, signal transducer and activator of transcription 3, and extracellular signal-regulated kinase-1/2 in injured kidneys. Collectively, these data suggest that MLL1 contributes to renal protection and functional recovery and promotes renal regeneration through a mechanism associated with activation of the epidermal growth factor receptor signaling pathway.NEW & NOTEWORTHY Mixed lineage leukemia 1 (MLL1) is a methyltransferase that induces histone H3 lysine 4 trimethylation and exerts its functional roles by interacting with multiple subunits. In this study, we demonstrated that inhibition of MLL1 activity by MI503 or MM102 aggravated renal injury and apoptosis and suppressed renal tubular cell dedifferentiation and proliferation, suggesting that MLL1 activation during acute kidney injury acts as an intrinsic protective mechanism to mediate renal tubular cell survival and regeneration.